Genome by Design

By: David Smoller, Biowire Volume 10 Article 1

Without a robust understanding of what is being observed, no conclusions can be drawn. In biology, the multifaceted nature of even basic in vitro systems makes this a complex task, requiring not just stringent control of as many variables as possible, but also an appreciation of the likely impact of those variables that cannot be influenced.

This is especially true in drug discovery, where a candidate compound that appears highly effective in biochemical trials may prove ineffectual, or even harmful, at a cellular or physiological level due to a wide variety of factors. Adverse drug reactions (ADRs) in particular can be highly detrimental for pharmaceutical manufacturers, either in terms of lost revenue or negative publicity, and generally stem from the inherent flaws in the traditional models routinely used for investigation of candidate drugs. Despite the wealth of knowledge gained from studying immortalized cell lines, for example, these only provide an approximation of the biology of the target organism.

To improve the accuracy of cell- and animal-based models, Sigma® Life Science is focused on development of innovative technologies – such as zinc finger nucleases – to allow highly accurate genetic manipulation of model cell lines and organisms, enabling researchers to answer specific biological questions.

Zinc finger nucleases (ZFNs) offer a unique solution for mammalian genome editing, and can be designed to generate site-specific double-strand breaks (DSBs) at virtually any point in the genome. These breaks can then be repaired by either non-homologous end joining (NHEJ), leading to gene knockout, or homologous recombination (HR), achieving precise gene insertion, deletion or replacement. This technique allows genes to be altered virtually at will, and has already been accomplished at multiple loci using ZFNs in conjunction with donor DNA containing the desired mutation. ZFNs also enable delicate, specific mutagenesis of single-base sites in the native context of the gene, lending more biological relevance to the resulting data. The ability to make such mutations enables a more rigorous investigation of single nucleotide polymorphisms (SNPs) associated with disease states. Application of ZFN technology to existing immortalized cell lines potentially offers several advantages for current cell-based assays, both in terms of introducing new genetic variations and eliminating erroneous mutations, however the advent of induced pluripotent stem cells (iPS cells) raises the possibility of developing bespoke cell lines which truly reflect genetic make-up of a given disease.

To truly understand the impact of editing the genome, and the subsequent effect of a candidate compound on negating this genetic change, it is necessary to go beyond basic cellular systems and investigate animal models. In the last 20 years, the availability of knockout mouse technology has established the mouse as the most widely used model system in biomedical research. The key to the success of this technology lies in the ease with which mouse embryonic stem (mES) cells can be cultured, genetically modified, cloned, and implanted into blastocysts to derive chimeric animals.

Although this technology provides an excellent mechanism for the study of basic mammalian genetics, it cannot be used to generate clinically-relevant data for drug discovery. However, in 2009, the first targeted knockout rats exploiting ZFNs were created via microinjection of ZFN mRNA into single cell embryos. Screening of the resulting animals was surprisingly rapid and efficient, enabling the production of founders in as little as two months post-microinjection, and involving screening of less than 100 animals. Most importantly, the ease of ZFN-mediated knockout rat generation indicates the suitability of the technique for targeted gene knockout in other species. Because the technique does not require ES cell lines for the species of interest, existing embryo isolation, injection and implantation protocols for creation of transgenic animals can be employed. As robust embryo manipulation methods already exist for a variety of species – including mice, rats, rabbits, chickens, sheep, goats, cows and pigs – this offers a straightforward route to efficiently delivering ZFNs and deriving knockout animals.

The advent of ZFN technology highlights the potential for such techniques to be used in development of new model systems to answer specific biological questions. To move forward our understanding of the potential physiological effects of candidate drugs, we need to embrace the complexity of living systems and develop new tools to deal with the immense quantities of information these models are able to generate. Greater throughput, coupled with the ability to collect more data per sample, will offer a more comprehensive look at how individual molecular interactions fit into the complex biochemical pathways of the living cell. The future is still dependent on scientists asking the right questions, but the ability to design and control new biological systems is an important step along the path to answering those questions.

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